Summary

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.

Introduction

After many decades of research and discussion, even the basic mechanisms behind the control of shoot branching remain obscure. The effects and interactions between various plant hormones, nutrients, water, light and gravity have all been investigated, but their precise roles are still unclear. Of the hormones believed to be involved, auxin has been the most extensively studied, and a great deal of physiological and molecular genetic evidence has accumulated to indicate that it plays a central role.

Much of the research into auxin and the control of shoot branching has centred on apical dominance. In many species, removal of the shoot apex results in the outgrowth of lateral branches (reviewed by Cline, 1991). Thimann and Skoog (1933) were the first to demonstrate that exogenous auxin, applied to the shoot stump of such decapitated plants, suppresses the outgrowth of lateral buds. A role for auxin in vivo is supported by the observation that application of auxin transport inhibitors to the stem of intact plants can reduce or abolish apical dominance ( Panigrahi and Audus, 1966; Snyder, 1949). Furthermore, the axr1 mutant of Arabidopsis has reduced auxin responses and increased lateral branching in both vegetative and floral nodes ( Lincoln et al., 1990 ; Stirnberg et al., 1999 ), transgenic plants with reduced auxin levels have increased branching ( Romano et al., 1991 ), and transgenic plants with increased auxin levels have reduced shoot branching ( Klee et al., 1987 ). These data strongly support the hypothesis that apically derived auxin is transported basipetally and inhibits the outgrowth of lateral buds. However, the mechanism by which auxin suppresses bud outgrowth is unknown. A direct effect is unlikely because experiments with radio-labelled indole-3-acetic acid (IAA) have shown that IAA transported basipetally from the shoot apex is not translocated acropetally into the inhibited lateral bud ( Hall and Hillman, 1975; Morris, 1977). Furthermore, auxin levels in the bud actually increase as lateral buds begin to enlarge and grow ( Gocal et al., 1991 ; Hillman, 1984; Pearce et al., 1995 ).

Several hypotheses have been proposed to explain how auxin might act indirectly to inhibit bud outgrowth. For instance, it has been suggested that auxin may induce the production of a secondary inhibitor, such as ethylene or abscisic acid (ABA), which then moves into the lateral bud. However, ethylene now appears to be an unlikely candidate for the secondary messenger. A study by Romano et al. (1993) , using transgenic tobacco and Arabidopsis plants that over-produced auxin while maintaining normal levels of ethylene, indicated that apical dominance was primarily controlled by auxin. In addition, Li and Bangerth (1992) could find no correlation between ethylene production and bud release in decapitated pea plants treated with four substances known to affect IAA, cytokinin and ethylene concentrations. The case for ABA is stronger because ABA levels have often been shown to correlate with bud dormancy ( Galoch et al., 1998 ; LeBris et al., 1999 ; Pearce et al., 1995 ; Piola et al., 1998 ); however, conclusive evidence regarding its role is still absent.

Another model proposes that auxin acts by regulating the export of cytokinin from the roots ( Bangerth, 1994). This model is based on the observations that cytokinin can directly promote the outgrowth of lateral buds (reviewed by Cline, 1991). Following removal of the shoot apex, cytokinin export from roots increases, and this increase can be blocked by application of auxin to the decapitated stump ( Bangerth, 1994). In an alternative model, Stafstrom (1993) proposes that gradients of the two hormones along the shoot axis (auxin transported basipetally, cytokinin moving acropetally from the roots) could be an important factor controlling the branching pattern. The phenotypes of transgenic plants with elevated levels of endogenous IAA or cytokinin certainly lend support to the proposed inhibitory and promotive roles in shoot branching (reviewed by Klee and Estelle, 1991; Klee and Romano, 1994). However, evidence from work with the rms mutants in pea demonstrates that the endogenous levels of these two hormones are not always correlated with the degree of shoot branching, leading to the suggestion that additional signals could be involved ( Beveridge et al., 1994 ; Beveridge et al., 1997a ; Beveridge et al., 1997b ). For example, it is suggested that auxin could act by directing the transport of nutrients away from lateral buds ( Croxdale, 1977; Patrick, 1979). However, the weight of experimental evidence does not support this model; particularly the observation that nutrients added directly to inhibited buds cannot promote outgrowth ( Phillips, 1975) .

It is clear from research to date that the interactions of the various plant hormones are complex and this makes it difficult to study their effects on lateral bud outgrowth in isolation. The need for close control of environmental and nutritional factors when analysing the effects of plant hormones on bud growth is also highlighted. In this paper, we describe an assay to examine the effects of basally and/or apically applied hormones on the growth of buds on excised nodal sections of Arabidopsis. The adoption of Arabidopsis as a system to study apical dominance allows the exploitation of a wide range of mutants to dissect the factors involved in regulating bud growth. By using nodal sections, we have tried to avoid some of the problems associated with the investigation of bud outgrowth in whole-plant experimental systems. In this paper, we have used hormone applications and hormone signalling mutants to test the roles of ethylene, cytokinin and abscisic acid in the inhibition of bud outgrowth by apical auxin.

Results

The experimental procedure presented here ( Figure 1) uses excised nodal sections embedded at each end in Arabidopsis thaliana salts (ATS) –agar nutrient media blocks. This gives a satisfactory growth rate for untreated buds and permits us to supply compounds to either side of the nodal section. The lowest node from the primary inflorescence (referred to as node 1) of sterile cultured Arabidopsis plants was excised with 6–10 mm of stem either side. These nodal sections were then used to bridge an 8 mm gap between two ATS–agar media blocks in a 9 cm Petri dish. The blocks on either side of the plate were amended with the required stock solution 72 h prior to the addition of the nodal sections. Compounds tested in this way included various plant hormones, phytotropins and metabolic inhibitors. An equivalent volume of the relevant solvent was added to control plates. The outgrowth of the bud on each nodal section was then measured every 24 h for the next 10–14 days.

Figure 1.

A diagram outlining the experimental procedure and showing the arrangement of Arabidopsis nodal sections within the split plates.

Growth of excised buds

In our first study, we compared the growth rates of buds on the excised nodal sections in our assay with those of axillary buds on the same node in intact plants. Growth of lateral shoots from excised node 1 was slow for the first 24 h (0–1 mm). The growth rate then increased steadily to settle at an average of 7 mm per day between 72 and 144 h ( Figure 2). The growth curve for lateral buds from node 1 on intact Columbia plants grown in sterile culture, under the same light and temperature regimes, was more linear than for the excised nodes ( Figure 2). Initially growth rates were similar, but although the growth rate of lateral shoots on excised nodes accelerated after 72 h, the comparative growth of lateral shoots on intact plants was significantly slower, attaining only 3.5 mm per day after 120 h.

Figure 2.

A comparison of the rates of lateral bud outgrowth from the lowest node of intact Arabidopsis plants (Columbia ecotype) and from the same node on excised untreated sections.

Data shown are means ± SE (n = 10).

The effect of auxin

1-naphthalene acetic acid (NAA, 1 µm) in the apical media block was found to inhibit lateral bud outgrowth for 4–7 days if the bud was no more than 1.5 mm in length at excision ( Figure 3a). Buds larger than 1.5 mm at excision of the nodal section were increasingly less likely to be inhibited by 1 µm NAA for any period, and most lateral buds greater than 3 mm in length grew out at rates not significantly different from control buds.

Concentrations of apical NAA higher than 1 µm prolonged the period of inhibition, with 10 µm inhibiting most buds for at least 7 days and many for the duration of the experiment: 10–14 days ( Figure 3b). NAA at this concentration (10 µm) could also inhibit outgrowth from longer buds, up to 3.5 mm long at excision (data not shown). At lower concentrations, inhibition was reduced. Apical 0.5 µm NAA inhibited the outgrowth of many buds, but usually for a shorter period than 1 µm NAA, and apical 0.1 µm NAA did not affect the rate of bud growth ( Figure 3b).

The most prevalent natural auxin, IAA, and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) supplied apically were also able to inhibit bud outgrowth ( Figure 3c). Apical IAA was found to be a less potent inhibitor than NAA. IAA at a concentration of 1 µm prevented outgrowth for 2–4 days and was similar in effect to 0.5 µm NAA. This observation may reflect the greater susceptibility of IAA to photo-oxidation in the medium, and to biodegradation or conjugation within the explants. Both NAA and IAA appeared to inhibit the lateral shoot for a period of time, after which growth proceeded at a rate comparable to the controls. By contrast, when apical 2,4-D was used, the buds were completely inhibited for 2–10 days and when growth began it proceeded at a greatly reduced rate.

When any of the auxins were supplied in the basal medium alone, bud outgrowth did not differ from controls even when a 10 µm concentration was used ( Figure 4a). To test whether this failure to inhibit lateral bud outgrowth might be due to the orientation of the basal media block and the node with respect to gravity, we set up split plates in which the nodes were positioned upside down. Even with the orientation of the nodes reversed, NAA supplied at the inverted apical end was found to inhibit outgrowth, but still had no effect when provided at the basal end (data not shown).

If 1 µm NAA was supplied both apically and basally simultaneously, the mean duration of inhibition, compared to apical NAA alone, was increased from 5 days to 7–8 days. NAA at a concentration of 10 µm supplied to both ends completely inhibited all outgrowth of the lateral shoots for the duration of the experiment (14 days). However, supplying 0.5 µm NAA at both ends resulted in bud outgrowth that was not significantly different from when 0.5 µm NAA was supplied only to the apical end (data not shown).

The effect of NPA on auxin-mediated inhibition

The observation that basal auxin alone does not affect outgrowth of the lateral shoots suggests that basipetal polar auxin transport may be important for inhibition by apical auxin in Arabidopsis. When the polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) was supplied apically (1 µm), inhibition by 1 µm apical NAA was partially relieved ( Figure 4b). By contrast, basal 1 µm NPA had no effect on inhibition. This indicates that polar auxin transport is required for inhibition by apical auxin. Application of NPA alone, either basally or apically, had a slight inhibitory effect on bud growth.

The effect of ethylene and ABA on auxin-mediated inhibition

A number of mechanisms have been proposed to account for the inhibition of lateral buds by apical auxin. Because auxin transported down the stem by basipetal polar auxin transport does not appear to accumulate in inhibited buds, it has been postulated that a secondary messenger is involved. Both ethylene ( Blake et al., 1983 ; Burg and Burg, 1968; Russel and Thimann, 1988) and ABA ( Tucker, 1980) have been put forward as possible candidates for this role. To determine whether ethylene is required for inhibition of bud outgrowth by apical auxin, experiments were carried out using the ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG) and the ethylene-insensitive mutant etr1 ( Figure 5a). The rate of lateral outgrowth in the etr1 mutant was slower than in wild-type, and 5 µm AVG, supplied in both media blocks, also reduced the rate of bud growth substantially. However, despite the lower growth rates, inhibition by apical 1 µm NAA was clearly demonstrated with both etr1 and AVG treatments. In fact, inhibition by apical 1 µm NAA was more prolonged when combined with either AVG or etr1 in the assay (24 h longer, on average, with AVG and 5–6 days longer with etr1). Hence ethylene biosynthesis and ethylene action do not mediate auxin inhibition of lateral bud growth.

Figure 5.

The roles of ethylene and ABA in auxin inhibition of lateral bud outgrowth.

Ethylene biosynthesis and ethylene action are not required for auxin inhibition of lateral bud outgrowth; buds on the ethylene-insensitive mutant etr1 and Columbia nodal sections treated with aminoethoxyvinylglycine (AVG) are inhibited by apical NAA (a). ABA-insensitive mutations abi1-1 and abi2-1 do not significantly alter the response of lateral buds to auxin (b). ABA alone at a concentration of 3 μm has little effect on the rate of lateral bud outgrowth (c). When combined with apical 1 μm NAA, the ABA enhances inhibition when supplied basally, reduces it when supplied apically, and has little effect when supplied to both sides (c). Outgrowth of abi1-1 buds is completely insensitive to 3 μm ABA, and auxin inhibition is unaffected (d). Data shown are means ± SE (n = 10–57).

To determine whether ABA signalling is required for auxin-mediated bud inhibition, the auxin responses of lateral buds from the ABA-insensitive mutants abi1-1 and abi2-1 were measured ( Figure 5b). Inhibition by apical NAA did not appear substantially different in either mutant compared with the wild-type (Landsberg erecta). These findings suggest that ABA is not acting as a secondary messenger for auxin inhibition of lateral bud outgrowth. It was also noted that buds on nodal sections from the Landsberg erecta ecotype grew out more slowly than those of Columbia, and hence the difference between auxin-treated and control buds is reduced.

The effect of ABA on the outgrowth of wild-type and ABA-insensitive mutant buds was also measured. Data collected for the Columbia ecotype using a 3 µm concentration of ABA, with and without apical 1 µm NAA, are shown in Figure 5(c). Basal and apical 3 µm ABA did not affect the rate of bud outgrowth significantly, but, when supplied in both media blocks, a slight reduction in growth rate was observed. Combining basal 3 µm ABA with apical 1 µm NAA increased the duration of inhibition significantly compared with apical NAA alone. Conversely, supplying both 3 µm ABA and 1 µm NAA apically resulted in a shortened period of inhibition and rapid bud outgrowth thereafter. ABA at a concentration of 3 µm on both sides and 1 µm NAA apically yielded a mean rate of bud growth midway between that observed for the basal and apical treatments. Broadly the same pattern of bud outgrowth responses was seen when a lower ABA concentration of 1.2 µm was used, except that the reduction in the duration of auxin inhibition by apical ABA became less pronounced (data not shown).

These experiments were repeated using Landsberg erecta and the ABA-insensitive mutant abi1-1. It was found that Landsberg erecta appeared generally more sensitive to ABA than Columbia. In Landsberg, the rate of bud outgrowth was dramatically reduced (and to approximately the same degree) by 3 µm ABA in any orientation, and, in contrast to Columbia, all combinations of ABA with apical 1 µm NAA resulted in much slower outgrowth than with apical 1 µm NAA alone. However, as in Columbia, buds to which ABA and NAA had been supplied together apically grew out sooner than those where basal ABA or ABA on both sides was combined with apical NAA (data not shown). When abi1-1 was tested, it was found that the buds were completely insensitive to ABA in any orientation and auxin inhibition was unaffected ( Figure 5d). This finding provides further evidence that ABA is not acting as a secondary messenger in auxin-mediated inhibition of bud outgrowth. It was also noted that 3 µm ABA in a media block tended to cause gradual yellowing of the Landsberg erecta nodal sections at the points of contact, but did not affect abi1-1 in this way.

The effect of cytokinin

Cytokinin has been widely reported to promote lateral bud outgrowth and to antagonize auxin inhibition (reviewed by Cline, 1991). To test the effects of cytokinin, the synthetic cytokinin benzyl adenine (BA) was added to either the apical, basal or both media blocks ( Figure 6a). Basal 1 µm BA caused a slight increase in the rate of lateral growth, such that buds were on average one-third longer than controls after 72 h. Apical cytokinin and cytokinin applied to both sides had little effect.

To investigate the interactions of auxin and cytokinin, different combinations of apical and basal NAA and BA were used ( Figure 6b). Basal 1 µm BA was found to release the inhibition of outgrowth by 1 µm apical auxin in most instances. However, the response to this combination was comparatively variable and a few buds remained inhibited, but for a shorter duration overall. When the orientation of the hormones was reversed, i.e. NAA basally and BA apically, the mean rate of lateral outgrowth was the same as the controls for the first 120 h, after which the buds from this treatment tended to die. When both hormones were applied basally, bud growth was similar to that of untreated controls. When cytokinin and auxin were supplied together apically, inhibition of bud outgrowth was greater than when auxin alone was used. Apical 1 µm NAA inhibited lateral bud growth for 4–7 days, but apical 1 µm NAA with 1 µm BA caused most buds to be inhibited for the duration of the experiment (10–14 days). Buds exposed to combined apical NAA plus BA were also more resistant to release by basal BA than buds exposed to apical NAA alone (data not shown).

Discussion

We have investigated the growth responses of buds on excised nodal sections of Arabidopsis to the plant hormones auxin, cytokinin, ethylene and ABA. Wild-type buds and buds from hormone-signalling mutants have been compared in order to investigate hormonal interactions in the control of bud growth. The assay we have developed allows various combinations of plant hormones and/or phytotropins and/or metabolic inhibitors to be supplied to each side of the node separately. We have demonstrated that the outgrowth of lateral buds from Arabidopsis can be inhibited by physiologically relevant concentrations of apical auxin. Previously, workers using decapitated Arabidopsis plants have been unable to restore apical dominance using exogenous auxin treatments ( Cline, 1996). The difference in response by buds on decapitated plants compared with buds on isolated nodes may be due to a number of factors. Perhaps the most attractive explanation is that the roots on the decapitated plants are supplying factors such as cytokinins, which counteract inhibition by the apically applied auxins.

Auxin and bud outgrowth

Our data show a direct relationship between the concentration of apical auxin and the duration of the inhibition of bud outgrowth ( Figure 3). Other workers have found that increasing the concentration of auxin applied to the cut stump of decapitated plants ( Thimann, 1937; Went, 1939), or to the apical end of explants ( Tamas et al., 1989 ), decreases the final length of lateral buds. Wickson and Thimann (1958) observed that buds on nodal sections of Pisum sativum immersed in nutrient solutions were inhibited for approximately the same period over a range of IAA concentrations. The buds would ‘breakaway’ after 5 days, but their subsequent growth was slower at higher IAA concentrations. In contrast, we have found that, when apical NAA is applied, the time to breakaway increases as the concentration is raised from 0.1 to 10 µm, often with little difference in the subsequent rate of outgrowth ( Figure 3b). The mechanism behind this concentration-dependent timing of breakaway is unclear. A decline in the sensitivity of some auxin-responsive tissues may occur, or the ability of the nodal sections to take up and/or transport auxin may diminish with time and the build-up of callus at the cut stump. Perhaps a substance accumulates that counteracts inhibition by auxin. For instance, cells at the base of the nodal sections that have begun to differentiate into root tissue may begin to synthesize and release cytokinins. We have established that there is no significant decrease in the potency of the NAA in the split plates during the course of the experiment. Plates used in the assay for 8 days were replenished with fresh nodal sections and were found to inhibit bud outgrowth for as long as fresh plates (data not shown).

The assay has also revealed a trend for decreasing sensitivity to inhibition by apical auxin as lateral bud size increases. We have found that some of the larger buds (2–5 mm), which appear unresponsive to 1 µm NAA, can be inhibited by higher concentrations of apical NAA, but that even 10 µm NAA may not inhibit buds longer than 5 mm (data not shown). Therefore, it appears that, with increasing size, buds loose competence to respond to auxin. The molecular basis for this change is unknown. One possibility is that the transition to auxin resistance corresponds to the establishment of auxin synthesis and transport in the developing bud as suggested by Snow (1937). Certainly, auxin levels increase as buds begin to grow ( Gocal et al., 1991 ; Hillman, 1984; Pearce et al., 1995 ), and bud activity correlates with the presence of active polar auxin transport ( Morris and Johnson, 1990; Morris, 1977).

Supplying apical NPA prevented inhibition by apical NAA. This supports the conclusion reached by Tamas et al. (1989) that basipetal auxin transport from the apex is required for bud growth inhibition. In addition, our observation that basally supplied auxins failed to inhibit lateral bud outgrowth is in agreement with their findings. However, they also found that supplying basal IAA to bean nodal explants could relieve inhibition by apical IAA, which is contrary to our results using NAA. This may reflect a difference in the action of these specific auxins (e.g. uptake into cells), or in the responses of the two species to basal auxin. The difference in the effects of basally and apically supplied auxin is not explained by a difference in the quantity of hormone reaching the bud. By the addition of dyes, we have established that there is significant bulk flow of solutes from the basal media block to the cauline leaf and later to the growing bud in the transpiration stream (data not shown). However, dye supplied in the apical media block did not appear in the leaf or bud. In addition, Lim and Tamas (1989) used 14C-IAA to show that the quantity of IAA transported to the bud from the cut apical surface of explants did not exceed that reaching the bud from the basal end. We were able to confirm this result for Arabidopsis explants using 14C-IAA in our own assay (data not shown).

The role of ethylene

The hypothesis that auxin might act via ethylene in the control of apical dominance ( Blake et al., 1983 ; Burg and Burg, 1968; Russel and Thimann, 1988) is not supported by recent research ( Cline, 1991; Li and Bangerth, 1992; Romano et al., 1993 ). Our own data, from experiments with AVG and the etr1 mutant, demonstrate that ethylene biosynthesis and ethylene action, respectively, are not required for inhibition of bud outgrowth by apical auxin. We therefore conclude that ethylene does not act as a second messenger for auxin in correlative inhibition of bud outgrowth.

The role of abscisic acid

There is a positive correlation between a reduction in ABA levels within buds of many species and the release of those buds from dormancy ( Galoch et al., 1998 ; LeBris et al., 1999 ; Pearce et al., 1995 ; Piola et al., 1998 ; reviewed by Cline, 1991). Further evidence for a role for ABA in regulating bud growth comes from analysis of the era1 mutant of Arabidopsis which has an enhanced response to ABA and reduced branching ( Pei et al., 1998 ). However, the relationship between ABA and auxin in the inhibition of bud outgrowth is unclear (reviewed by Cline, 1991). Our data do not support a model in which auxin acts through ABA to inhibit bud growth. Buds from the ABA-insensitive mutants abi1 and abi2 have a wild-type response to apical NAA in the assay. In addition, abi1 buds did not respond to 3 µm ABA in any orientation, alone or in the presence of apical NAA. Therefore, ABA signalling appears not to be required for auxin to inhibit bud growth. Our data are consistent with an independent role for ABA in regulating bud growth.

We have shown that application of ABA can significantly alter the duration and character of inhibition by apical auxin in a manner dependent upon the end of the nodal section to which it is applied. Apical ABA can reduce auxin-mediated inhibition, whilst basal ABA enhances it. It is possible that these effects reflect natural processes such that, depending on where ABA is synthesized and/or how it is transported and/or the tissues it reaches, ABA in vivo may either promote or relieve inhibition by auxin. However, it is also possible that one or more of the observed responses to ABA may be non-physiological, due perhaps to the concentration of the mixed isomers used and/or contact with ectopic tissues. For example, one attractive hypothesis is that the normal role of ABA in dormant buds is to block the establishment of polar auxin transport in the bud. Consistent with this idea, as discussed above, a reduction in ABA levels and an increase in polar auxin transport in the bud both correlate with release from dormancy. If this hypothesis is correct, then ABA applied ectopically to the apical end of the excised node could block the action of apically applied auxin by preventing its loading into the polar transport stream. In contrast, basally applied ABA could be drawn into the bud by transpiration and enhance bud dormancy by inhibiting the establishment of polar auxin transport in the bud.

The role of cytokinin

The cytokinin BA, when applied basally, apically or both, had relatively little effect on the outgrowth of the lateral buds in the assay, although a small stimulation of the rate of growth was seen when 1 µm BA was added basally. Similarly, Wickson and Thimann (1958) found that kinetin alone did not have a marked growth-promoting effect on the lateral buds of Pisum stem sections. Shein and Jackson (1972) and Davies et al. (1966) both found that kinetin applied alone to the cut stump of decapitated plants did not alter the rate of bud outgrowth significantly compared with controls.

Cytokinins have been shown to stimulate bud outgrowth when applied to the lateral buds of plants with an intact apex, or to decapitated plants to which inhibitory concentrations of apical auxin were applied ( Mok, 1994). In plants, the roots are believed to be the major site of cytokinin synthesis ( Chen et al., 1985 ), and to export these substances to the shoot via the xylem. The export of cytokinins from roots has been shown to be influenced by apical auxin, and this has led to the hypothesis that inhibition by apical auxin is mediated wholly or partly by its influence on cytokinin export from the roots ( Bangerth, 1994). Our data are not consistent with this hypothesis because auxin is clearly able to inhibit bud growth in the absence of roots. A conceptually similar model is suggested by recent work, which indicates that auxin may suppress cytokinin biosynthesis ( Eklof et al., 1997 ; Kaminek et al., 1997 ). In this case, apical auxin acts by suppressing cytokinin synthesis in the stem and hence reduces the level of cytokinin reaching the bud. At this stage it is impossible to distinguish between this and a more classical model, similar to that proposed by Stafstrom (1993), in which opposing auxin and cytokinin gradients control shoot branching; with the auxin:cytokinin ratio being the critical factor. Consistent with this idea, we found that increasing the basal BA:apical NAA ratio further reduced auxin inhibition of lateral buds in the assay (data not shown).

The effect of apical cytokinin is more difficult to interpret in a physiological context. In our assay, apically applied cytokinin enhanced the effect of apical auxin. These results are consistent with previous studies showing that applying cytokinin together with auxin to the cut stem of decapitated plants can enhance auxin inhibition of lateral bud growth ( Ali and Fletcher, 1971; Davies et al., 1966 ; Hillman, 1970; Phillips, 1969). Evidence suggests that cytokinin applied to the cut stump or apical bud may strengthen auxin inhibition by enhancing IAA transport from the apex of plants ( Davies et al., 1966 ; Li and Bangerth, 1992). Kinetin has also been found to enhance the movement of 32P and 14C-sucrose towards the cut stump to which IAA has been applied ( Seth and Wareing, 1964). The interaction of these hormones therefore appears to be complex.

Conclusion

The assay that we have developed, combined with the wide range of Arabidopsis mutants available, is proving a valuable method for investigating the roles of different hormones in the inhibition and outgrowth of lateral buds in Arabidopsis. We have confirmed previous studies demonstrating that apically derived auxin travelling in the polar transport stream inhibits the outgrowth of lateral buds. Small buds are more sensitive to apical auxin than large buds, which become essentially unresponsive to the hormone. In addition, we have found that auxin inhibition appears to be independent of ethylene and ABA signalling, and can be over-ridden by basally applied cytokinin and enhanced by basally applied ABA. In general, our data are less supportive of models in which ethylene, ABA or cytokinin act directly downstream of auxin in regulating branching. Rather they favour models in which these hormones are acting independently to regulate bud activity. Thus, it appears that the second messenger relaying the auxin signal into the bud is, with the possible exception of cytokinin, unlikely to be any of the hormonal candidates.

We are now using the assay to assess the effect of altering the availability of various nutrients on lateral bud outgrowth and auxin inhibition. It is hoped that by manipulating the levels of these factors within the two media blocks, insight can be gained into their contributions to the regulation of lateral bud outgrowth. Combining these treatments and techniques with the substantial genetic resources available in Arabidopsis will allow a detailed analysis of the control of bud outgrowth.

Experimental procedures

Plant material and growth conditions

Arabidopsis thaliana plants of the Columbia ecotype were used to provide the excised nodal sections, except for work with abi1-1 and abi2-1 where Landsberg erecta served as wild-type. The etr1 mutant line was supplied by the Nottingham Arabidopsis Stock Centre and abi1-1 and abi2-1 were provided by Peter McCourt (University of Toronto, Toronto, Canada).

Excising the nodes

Plants were selected as they developed to an appropriate point for excision of the nodal sections, typically between 20 and 27 days after sowing. Nodes were excised when sufficient internode was accessible to allow 6–10 mm of stem either side of a node, but before the axillary bud had grown to more than 1.5 mm in length. These conditions were most often satisfied for the lowest node, i.e. the first cauline leaf above the rosette (node 1). All of the data displayed in this paper represent the growth responses of lateral buds from node 1. Although the rate of growth of lateral buds from these lowest nodes was sometimes slow (particularly with nodes separated from the rosette by a short internode), it was difficult to collect enough nodes higher up the primary inflorescence with sufficiently long internodes before their buds had started elongating rapidly.

Under sterile conditions, individual plants were removed from the jars and nodes excised with a scalpel. Two nodes were then transferred to one prepared ‘split plate’ using fine tweezers tipped with foam rubber. They were positioned so that at least 2 mm of the tip of the cut stem on each side was embedded in the media. This operation was performed as quickly as possible to avoid desiccation of the excised section. Figure 1 shows how the nodes were positioned in the plates.

Split plate production

Standard Petri dishes (9 cm) were filled with 24.5 ml of ATS and left to set in a laminar flow hood for 45–60 min. An 8 mm wide strip of the medium could then be removed from the centre of the plate. This trough was checked carefully for any bridges between the two media blocks and dried in the laminar flow hood, parallel to the airflow, for a further 30 min.

When the plates were dry, a micro-pipette was used to inject 5–20 µl of solutions of various compounds into one or both of the separated media blocks at standardized points. The initial volume of the media blocks on either side was 10.5 ± 0.25 ml each. However, water was lost from the plates as they dried over the course of an experimental run, so the quantity of hormone stock solution to be added was calculated assuming a volume of 10 ml for each block. The plates were then left at 4°C for at least 72 h to allow the hormone to diffuse evenly throughout the media.

Stock solutions consisted of a 1, 2 or 10 m m concentration of each of the various compounds dissolved in 70% ethanol. Stocks were stored at −20°C and fresh solutions were made up every month. ABA (mixed isomers, Sigma A7383) was dissolved in methanol (5% of complete stock solution) then mixed with ethanol (65%) and sterile distilled water (30%) and stored at −20°C. Fresh ABA stock was prepared every 10 days.

Set-up and measurements

The split plates were held in wire racks 10° off vertical, with the nodes upright. They were placed in the same chamber under the same growing conditions as the original plants. Growth of the lateral buds was then measured every 24 h (for 10–14 days) by aligning a ruler behind the plate. As they grew up vertically, the angle of the plates allowed most of the buds to pass over the media block in which the apical end of the excised stem was embedded. Individuals were excluded from the data set if the growing bud had touched the medium, if the cut end of the internode had been pushed out of the medium by elongation of the stem, if condensation may have bridged the gap between the two media blocks, or where microbial contamination was visible on the plate.

Acknowledgements

We thank Celina Jackson, Pamela MacKay, Emma Ronald and Edwin Lloyd-Jones for their technical assistance; the horticultural team for plant care; Dr Jon Booker, Dr Hanma Zhang, Dr Philip O'Donnell and Dr Sean Simpson for helpful discussions; Stephen Day for critical reading of the manuscript, and all those in the plant laboratory who at one time or another assisted with bud measuring. This research was funded by the Biotechnology and Biological Sciences Research Council .